Pieter Goossens

Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

Myeloid Type I Interferon Signaling Promotes Atherosclerosis by Stimulating Macrophage Recruitment to Lesions

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis

Atherosclerosis is a lipid‐driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth‐derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow‐derived macrophages, SEAs induce anti‐inflammatory macrophages, typified by high levels of IL‐10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR‐/‐ mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS‐injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T‐lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6Chigh monocytes, and macrophages showed high IL‐10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA‐treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF‐α, monocyte chemotactic protein 1 (MCP‐1), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth‐derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.—Wolfs, I. M. J., Stöger, J. L., Goossens, P., Pöttgens, C., Gijbels, M. J. J., Wijnands, E., van der Vorst, E. P. C., van Gorp, P., Beckers, L., Engel, D., Biessen, E. A. L., Kraal, G., van Die, I., Donners, M. M. P. C., de Winther, M. P. J. Reprogramming macrophages to an anti‐inflammatory phenotype by helminth antigens reduces murine atherosclerosis. FASEB J. 28, 288–299 (2014). www.fasebj.org

Macrophage Heterogeneity: Relevance and Functional Implications in Atherosclerosis

Atherosclerosis is a chronic inflammatory disease involving many cell types with a well-accepted key role for macrophages. A wide array of different properties and functional characteristics are attributed to macrophages present in the atherosclerotic plaque. As an increasing body of evidence strengthens the consensus that macrophages comprise a heterogeneous population, several co-existing subtypes with diverse, even opposing specialties have already been described in fields like parasitology, tumour biology and metabolic disorders. However, macrophage heterogeneity within atherosclerotic lesions has not been studied in detail yet. In this review we will introduce the characteristics of macrophage subsets in other pathologies and address the presence and possible roles of distinct macrophage subtypes in the rapidly evolving field of atherosclerosis. Finally, we make an effort to relate these subtypes to disease progression and explore a number of opportunities for novel diagnostic and therapeutic approaches.

Myeloid IκBα deficiency promotes atherogenesis by enhancing leukocyte recruitment to the plaques

Activation of the transcription factor NF-κB appears to be involved in different stages of atherogenesis. In this paper we investigate the role of NF-κB inhibitor IκBα in atherosclerosis. Myeloid-specific deletion of IκBα results in larger and more advanced lesions in LDL-R-deficient mice without affecting the compositional phenotype of the plaques or systemic inflammatory markers in the plasma. We show that IκBα-deleted macrophages display enhanced adhesion to an in vitro endothelial cell layer, coinciding with an increased expression of the chemokine CCL5. Also, in vivo we found that IκBαdel mice had more leukocytes adhering to the luminal side of the endothelial cell layers that cover the atherosclerotic plaques. Moreover, we introduce ER-MP58 in this paper as a new immunohistochemical tool for quantifying newly recruited myeloid cells in the atherosclerotic lesion. This staining confirms that in IκBαdel mice more leukocytes are attracted to the plaques. In conclusion, we show that IκBα deletion in myeloid cells promotes atherogenesis, probably through an induced leukocyte recruitment to plaques.

IFN-γ priming of macrophages represses a part of the inflammatory program and attenuates neutrophil recruitment

Macrophages form a heterogeneous population of immune cells, which is critical for both the initiation and resolution of inflammation. They can be skewed to a proinflammatory subtype by the Th1 cytokine IFN-γ and further activated with TLR triggers, such as LPS. In this work, we investigated the effects of IFN-γ priming on LPS-induced gene expression in primary mouse macrophages. Surprisingly, we found that IFN-γ priming represses a subset of LPS-induced genes, particularly genes involved in cellular movement and leukocyte recruitment. We found STAT1-binding motifs enriched in the promoters of these repressed genes. Furthermore, in the absence of STAT1, affected genes are derepressed. We also observed epigenetic remodeling by IFN-γ priming on enhancer or promoter sites of repressed genes, which resulted in less NF-κB p65 recruitment to these sites without effects on global NF-κB activation. Finally, the epigenetic and transcriptional changes induced by IFN-γ priming reduce neutrophil recruitment in vitro and in vivo. Our data show that IFN-γ priming changes the inflammatory repertoire of macrophages, leading to a change in neutrophil recruitment to inflammatory sites.

Deleting myeloid IL-10 receptor signalling attenuates atherosclerosis in LDLR-/- mice by altering intestinal cholesterol fluxes

Inflammatory responses and cholesterol homeostasis are interconnected in atherogenesis. Interleukin (IL)-10 is an important anti-inflammatory cytokine, known to suppress atherosclerosis development. However, the specific cell types responsible for the atheroprotective effects of IL-10 remain to be defined and knowledge on the actions of IL-10 in cholesterol homeostasis is scarce. Here we investigated the functional involvement of myeloid IL-10-mediated atheroprotection. To do so, bone marrow from IL-10 receptor 1 (IL-10R1) wild-type and myeloid IL-10R1-deficient mice was transplanted to lethally irradiated female LDLR-/- mice. Hereafter, mice were given a high cholesterol diet for 10 weeks after which atherosclerosis development and cholesterol metabolism were investigated. In vitro, myeloid IL-10R1 deficiency resulted in a pro-inflammatory macrophage phenotype. However, in vivo significantly reduced lesion size and severity was observed. This phenotype was associated with lower myeloid cell accumulation and more apoptosis in the lesions. Additionally, a profound reduction in plasma and liver cholesterol was observed upon myeloid IL-10R1 deficiency, which was reflected in plaque lipid content. This decreased hypercholesterolaemia was associated with lowered very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) levels, likely as a response to decreased intestinal cholesterol absorption. In addition, IL-10R1 deficient mice demonstrated substantially higher faecal sterol loss caused by increased non-biliary cholesterol efflux. The induction of this process was linked to impaired ACAT2-mediated esterification of liver and plasma cholesterol. Overall, myeloid cells do not contribute to IL-10-mediated atheroprotection. In addition, this study demonstrates a novel connection between IL-10-mediated inflammation and cholesterol homeostasis in atherosclerosis. These findings make us reconsider IL-10 as a beneficial influence on atherosclerosis.

Heterogeneity of atherosclerotic plaque macrophage origin, phenotype and functions : Implications for treatment

Abstract Macrophages are key players in atherosclerotic lesions, regulating the local inflammatory milieu and plaque stability by the secretion of many inflammatory molecules, growth factors and cytokines. Monocytes have long been considered to be the main source of plaque macrophages. However, recent findings provide evidence for proliferation of local macrophages or transdifferentiation from other vascular cells as alternative sources. Recent years of research focused on the further identification and characterisation of macrophage phenotypes and functions. In this review we describe the advances in our understanding of monocyte and macrophage heterogeneity and its implications for specific therapeutic interventions, aiming to reduce the ever growing significant risk of cardiovascular events without any detrimental side effects on the patients immune response.